Botulinum toxin prefilled plastic syringe

ABSTRACT

The present invention relates to a prefilled plastic container, such as a plastic syringe, comprising an aqueous botulinum toxin formulation. The aqueous botulinum toxin formulation in the prefilled plastic container is stable for a prolonged time period. Furthermore, the present invention relates to a kit comprising the prefilled plastic container, and to the use of the prefilled plastic container for therapeutic and cosmetic purposes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage entry of International ApplicationNo. PCT/EP2015/002600, filed Dec. 22, 2015, which claims priority toEuropean Patent Application No. 15000310.1, filed Feb. 3, 2015.

BACKGROUND Field

The present invention relates to a prefilled plastic container, such asa prefilled plastic syringe, comprising an aqueous botulinum toxinformulation. The aqueous botulinum toxin formulation in the prefilledplastic container is stable for a prolonged time period. Furthermore,the present invention relates to a kit comprising the prefilled plasticcontainer, and to the use of the prefilled plastic container fortherapeutic and cosmetic purposes.

Description of Related Art

Botulinum toxin (BoNT) is one of the most potent toxins known and actsby blocking acetylcholine release at peripheral cholinergic neurons.BoNT is synthesized as a 150 kDa precursor neurotoxic polypeptide and isactivated by selective proteolytic cleavage to yield the activetwo-chain BoNT form consisting of a 100 kDa heavy chain (HC; includesthe translocation domain and receptor-binding domain) and a 50 kDa lightchain (LC; includes the catalytic domain) linked by a disulfide bond andnon-covalent interactions. There are eight homologous serotypes (A, B,C₁, C₂, D, E, F, and G) of botulinum toxin, which are produced by thebacterium Clostridium botulinum in the form of a complex consisting of aneurotoxic polypeptide and other (non-toxic) clostridial proteins (i.e.different hemagglutinins and a nontoxic, non-hemagglutinating protein).

Careful administration of very small doses of toxin can restrict itsaction locally to reduce overactive muscles and exocrine glands.Therefore, botulinum toxin is now used in the treatment of a wide rangeof debilitating neuromuscular diseases (e.g., cervical dystonia,blepharospasm, and spasticity), overactive exocrine glands (e.g.,hyperhidrosis and hypersalivation) and other disease as well as foraesthetic purposes (e.g., treatment of facial wrinkles).

Botulinum toxins are inherently instable and, in particular, are knownto be highly unstable at alkaline pH and heat-labile. Additionally, itis known that dilution of the isolated toxin complex from milligramquantities to the much lower toxin concentrations used in solutions forinjection (in the nanograms per milliliter range) presents significantdifficulties because of the rapid loss of specific toxicity upon suchgreat dilution. This leads to loss of biological activity duringproduction, reconstitution and/or storage of protein-containingpharmaceutical compositions. These problems observed with proteins maybe due to chemical instability, resulting in bond formation or cleavage(e.g., hydrolysis, oxidation, racemization, β-elimination and disulfideexchange), and/or due to physical instability of the second orhigher-order structure of proteins without covalent bond-breakingmodification (e.g., denaturation, adsorption to surfaces, andnon-covalent self-aggregation).

The stability of pharmaceutical products is, however, of paramountimportance to ensure safe and efficacious use for a sufficiently longtime period. Since aqueous botulinum toxin formulations are particularlyprone to degradation, commercial preparations of botulinum toxin oftencome as vacuum-dried or lyophilized material. Examples include, forexample, Botox® (onabotulinumtoxinA; Allergan, Inc.) and Dysport®(abobotulinumtoxinA; Ipsen Ltd.), which both contain the C. botulinumtoxin complex of type A. Another example is Xeomin® (incobotulinumtoxin;Merz Pharma GmbH & Co. KGaA), which contains the pure neurotoxiccomponent of serotype A (i.e. the 150 kDa neurotoxic polypeptide) and isdevoid of any other proteins of the Clostridium botulinum toxin complex(i.e. the different hemagglutinins and the nontoxic,non-hemagglutinating protein).

However, while the lyophilized material has an increased stability, ithas generally to be reconstituted with a pharmaceutically acceptableliquid (e.g., saline) prior to use. Lyophilized pharmaceutical productsare therefore considered to be less convenient than other dosage forms.Also, the reconstitution process entails the risk of mismanagementresulting in inaccurate dosing or sterility issues. In addition, thelyophilization process is time-consuming and results in additionalcosts.

Another disadvantage of reconstituted solutions of botulinum toxin isthat they are often not entirely used because not every patient andindication requires the same dosage. Unfortunately, due to itsinstability, the reconstituted toxin solution can only be stored andre-used for a relatively short period. For example, after dilution withnormal saline prior to use, Botox® and Dysport® are recommended to beused within 6 hours and 4 hours, respectively. Likewise, the packageleaflet of Xeomin® specifies that after storage for more than 24 hours,the reconstituted Xeomin® solution shall no longer be used.

A medical dosage form which overcomes most of these disadvantages is theprefilled syringe format, which has been become increasingly popular inrecent years as drug delivery device. However, if proteins are used asactive ingredients, the limited stability of proteins renders it aparticularly difficult task for formulation scientists to use aprefilled syringe format. In particular, this applies to very diluteaqueous botulinum toxin solutions.

In order to increase stability of solid or liquid pharmaceuticalbotulinum toxin compositions, stabilizing proteins such as human serumalbumin (HSA) are often added. Also, it is known to addnon-proteinaceous stabilizing agents, such as surfactants,polyvinylpyrrolidone (PVP), disaccharides, polyols and the like.However, stability of liquid botulinum toxin formulations is stillunsatisfactory and/or is achieved using undesirable substances for humanuse by injection (see, e.g., WO 01/58472, WO 2006/005910, and WO2007/041664).

Furthermore, a liquid formulation of highly concentrated botulinum toxintype B (about 2500 U/ml) that is stable when stored in glass vials at 5°C. for up to 30 months is disclosed in WO 00/15245. However, thisstability is only achieved by using vials made of glass and bufferingthe pH of the solution down to an acidic pH of between 5 and 6, whichcauses pain upon injection.

Despite the advancements in the art, there is still no injectablebotulinum toxin presentation available which is not only stable over along period to provide a sufficiently long shelf life, but is alsoconvenient and easy to use, reduces medication errors, and minimizes therisk of contamination.

OBJECTIVE OF THE INVENTION

In view of the above, the objective of the present invention is toprovide a stable medical dosage form for the administration of botulinumtoxin in a convenient, safe and simple manner.

SUMMARY OF THE INVENTION

The above object is solved by the provision of a botulinum toxinprefilled plastic container (e.g., a syringe, vial, carpule or ampoule).The liquid botulinum toxin formulation in the prefilled plasticcontainer (e.g., a syringe, vial, carpule or ampoule) is stable at 2-8°C. over a prolonged period of time to provide a sufficiently long shelflife (at least about 12-24 months).

In a first aspect, the present invention provides a prefilled plasticcontainer (e.g., a syringe, vial, carpule or ampoule) comprising anaqueous botulinum toxin formulation, wherein the toxin activity is notreduced by more than 25%, relative to the initial toxin activity, uponstorage of the prefilled container for (a) 12 months at 5° C. or (b) 3months at 25° C.

The stability of the aqueous botulinum toxin formulation in theprefilled container (e.g., a syringe, vial, carpule or ampoule) in termsof the count (number) of sub-visible particles equal to or greater than10 μm is also excellent and generally below 1000/ml during storage for 6to 24 months (e.g., 6, 9, 12, 15, 18 or 24 months) at 2-25° C. (e.g., at5° C. or 25° C.). Furthermore, the aqueous botulinum toxin formulationin the prefilled container exhibits an excellent pH stability asindicated by a pH value that is generally not increased or decreased bymore than 10%, relative to the initial pH value, during storage of theprefilled container (e.g., a syringe, vial, carpule or ampoule) for 6 to24 months (e.g., 6, 9, 12, 15, 18 or 24 months) at 2-25° C. (e.g., at 5°C. or 25° C.).

In another aspect, the present invention provides a kit comprising aprefilled plastic container (e.g., a syringe, vial, carpule or ampoule)according to the first aspect of the invention and, optionally,instructions for use of said prefilled plastic container.

In a further aspect, the present invention provides a prefilled plasticcontainer (e.g., a syringe, vial, carpule or ampoule) according to thefirst aspect of the present invention for use in therapy. For example,the prefilled plastic container (e.g., a syringe, vial, carpule orampoule) may be used for treating a disease or condition caused by orassociated with hyperactive cholinergic innervation of muscles orexocrine glands in a patient including, but not limited to, dystonia,spasticity, paratonia, diskinesia, focal spasm, strabismus, tremor,tics, migraine, sialorrhea and hyperhidrosis.

In still another aspect, the present invention relates to the use of theprefilled plastic container (e.g., a syringe, vial, carpule or ampoule)according to the first aspect of the invention in cosmetic applications,such as for treating wrinkles of the skin and facial asymmetries, e.g.glabellar frown lines, crow's feet, upper facial rhytides and platysmabands.

In a yet further aspect, the present invention provides a method for thetreatment of a disease or condition caused by or associated withhyperactive cholinergic innervation of muscles or exocrine glands in apatient, the method comprising locally administering an effective amountof botulinum toxin to a muscle or exocrine gland of the patient byinjection using the prefilled plastic container (e.g., a syringe, vial,carpule or ampoule) according to the first aspect of the invention.

In a still further aspect, the present invention relates to a method forthe cosmetic treatment of the skin, such as for treating wrinkles of theskin and facial asymmetries, the method comprising locally administeringan effective amount of botulinum toxin to a patient by intradermal,subdermal or subcutaneous injection using the prefilled plasticcontainer (e.g., a syringe, vial, carpule or ampoule) according to thefirst aspect of the present invention.

Further embodiments of the present invention are set forth in theappended dependent claims. The present invention may be more fullyunderstood by reference to the following detailed description of theinvention, the examples and the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the stability of a liquid botulinum toxin formulation inprefilled syringe configurations A (●) and B (∘) at 5° C. as a functionof toxin potency versus time.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is based on the unexpected finding that a liquidbotulinum toxin formulation in a plastic container (e.g., in the form ofa syringe, vial, carpule or ampoule) shows an excellent long-termstability at reduced temperatures (e.g., 2-8° C.). Even upon storage atambient temperature (e.g., 25° C.) the botulinum toxin prefilledcontainer exhibits a surprisingly high stability.

Accordingly, the liquid botulinum toxin formulation prefilled containerof the present invention, in particular the botulinum toxin prefilledsyringe of the present invention, not only has a sufficiently long shelflife (at least about 12-24 months), but presents several additionaladvantages in comparison to other administration forms, such as easy andconvenient use, reduced risk of medication errors, high dosing accuracy,low risk of contamination, improved sterility assurance, and high safetyin administration.

Furthermore, the use of plastic materials for containers (e.g.,syringes) offers advantages over glass containers (e.g., glass syringes)in terms of break resistance, decreased weight, increased flexibilityfor novel shapes of primary containers, improved dimensional tolerances,and absence of undesirable substances (e.g., adhesives).

Plastic materials contain various substances and additive (e.g.,plasticizer) commonly referred to as leachables/extractables that areknown to easily destabilize proteins, in particular if they are of afragile nature and/or used at such low concentrations as neurotoxins(e.g., botulinum toxin). Therefore, liquid neurotoxin pharmaceuticalformulations are conventionally injected using glass syringes.Surprisingly, however, the prefilled plastic container (e.g., syringe)according to the present invention was found to provide stability to anaqueous botulinum toxin formulation for a long storage time (at leastabout 12-24 months) at 2-8° C. and, thus, provides a sufficiently longshelf life.

As used herein, a “prefilled container” refers to any device having apartially or fully enclosed space that can be sealed or is sealed andcan be used to contain, store, and/or transport liquid formulations. A“prefilled container” within the meaning of the present invention ispreferably a closed (or sealed) container made of, or partially orpredominantly made of, plastic (e.g., organic polymers) and includes,for example, containers in the form of (i) a syringe, (ii) a vial, (iii)a carpule, or (iv) an ampoule.

Prefilled syringes and carpules have two openings that are sealed toprevent leakage of the contents (e.g., aqueous formulations). In case ofa prefilled syringe, the proximal end is sealed by a plunger stopper andthe distal end is sealed by a capping device, as explained in detailherein below. In case of a plastic carpule, which is generally a plasticcylinder sterile filled with a drug formulation, the proximal end istypically sealed by a rubber stopper. This rubber stopper can be pressedin as a piston in the cylinder by the pressure of a punch of the carpulesyringe. The distal end is typically sealed by a puncture membrane. Thepuncture membrane is pierced for injection.

A “vial” within the meaning of the present invention is a vessel, whichhas usually a tubular form or a bottle-like shape with a neck and issuitable for containing, storing, and/or transporting drug formulations.The single opening is sealable by different vial closure systems. Forexample, vials may be closed with a screw cap (screw vials), a stopperof cork, plastic or rubber (lip vials and crimp vials) and other closuresystems like flip-tops or snap caps. Within the present invention a“vial” preferably means a plastic vessel having its opening sealed witha vial closure system.

In the following, the present invention is described in more detail. Itis pointed out that, although the term “prefilled syringe”, “prefilledplastic syringe”, “syringe” or “plastic syringe” is used in the detaileddescription of the invention, this does not mean that it is limited to a(plastic) syringe as a particular embodiment of the (plastic) container.In fact, any reference herein to a “prefilled syringe”, “prefilledplastic syringe”, “syringe”, “plastic syringe” or the like is to beunderstood as a reference to, and disclosure of, a “container” or“plastic container” and also includes, or discloses, a “vial” or“plastic vial”, a “carpule” or “plastic carpule”, or an “ampoule” or“plastic ampoule”, unless otherwise stated.

In a first aspect, the present invention relates to a prefilled plasticsyringe comprising botulinum toxin in an aqueous formulation, whereinthe toxin activity is not reduced by more than 25%, relative to theinitial toxin activity, upon storage of the prefilled syringe for (a) 12months at standard refrigerator temperatures (i.e. 2-8° C., such as 5°C.), or (b) 3 months at 25° C. Preferably, the toxin activity is notreduced by more than 20%, 15%, 10% or 5%, relative to the initial toxinactivity, upon storage of the prefilled syringe for 12 months at 2-8° C.(e.g., 5° C.) or is not reduced by more than 20%, relative to theinitial toxin activity, upon storage of the prefilled syringe for 3months at 25° C. Further preferably, the toxin activity is not reducedby more than 15%, 10% or 5%, relative to the initial toxin activity,upon storage of the prefilled syringe for 6 months at 2-8° C. (e.g., 5°C.).

Surprisingly, the aqueous botulinum toxin formulation in the prefilledsyringe is also stable for even longer storage times of up to 24 months.For example, upon storage for up to 24 months (e.g., 15, 18 or 24months) at 2-8° C. (e.g., 5° C.), the toxin activity is preferably notreduced by more than 30% or 25%, more preferably by no more than 20%, inparticular by no more than 15%, particularly preferable by no more than10%, and most preferable by no more than 5%, relative to the initialtoxin activity.

Within the present invention, the term “toxin potency” broadly refers toa measure of drug (e.g., botulinum toxin) activity expressed in terms ofthe amount required to produce an effect of a given intensity. The term“activity” or “toxin activity”, as used herein, refers to the biologicalactivity of the botulinum toxin, wherein “biological activity” may referto (a) receptor binding, (b) internalization, (c) translocation acrossthe endosomal membrane into the cytosol, and/or (d) endoproteolyticcleavage of proteins involved in synaptic vesicle membrane fusion. Forexample, any light chain (LC) domain, which shows proteolytic activityof more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and up to 100%of the corresponding wild-type LC domain in a SNAP-25 assay may beconsidered “biological active” or “to exhibit proteolytic activity”within the scope of this invention. Furthermore, any heavy chain (HC)domain that is capable of binding to a cellular HC domain receptor, inparticular to its native HC domain receptor, and is capable oftranslocating an LC domain attached to it, is considered “biologicallyactive”.

The biological activity is commonly expressed herein in Mouse Units(MU). As used herein, 1 MU is the amount of neurotoxic component, whichkills 50% of a specified mouse population after intraperitonealinjection, i.e. the mouse i.p. LD₅₀, as measured in accordance with themethod of Schantz and Kauter (Schantz and Kauter, J. Assoc. Off. Anal.Chem. 1978, 61:96-99). The terms “MU” and “Unit” or “U” are usedinterchangeable herein.

Suitable assays for determining the potency or biological activityinclude the mouse hemidiaphragm assay (MHA) described by Pearce et al.(Toxicol. Appl. Pharmacol. 128: 69-77, 1994) and Göschel et al. (Exp.Neurol. 147:96-10, 1997), the mouse diaphragm assay (MDA) according toDressler et al. (Mov. Disord. 20:1617-1619, 2005), a SNAP-25 proteaseassay (e.g., the “GFP-SNAP25 fluorescence release assay” described in WO2006/020748 or the “improved SNAP25 endopeptidase immuno-assay”described in Jones et al., J. Immunol. Methods 329:92-101, 2008), theelectrochemoluminescence (ECL) sandwich ELISA described in WO2009/114748, and cell-based assays as those described in WO 2009/114748,WO 2004/029576, WO 2013/049508 and WO 2014/207109.

As used herein, the term “initial toxin activity” or “initial toxinpotency” generally refers to the activity of the botulinum toxin at thebeginning of the storage period, i.e. after manufacture of the finalbotulinum toxin prefilled syringe, e.g. one week or less aftermanufacture. Further, the term “upon storage”, as used herein, isintended to mean after or at the end of storage for a given time period.In addition, the term “during storage” generally means over the courseof the entire storage period.

Apart from a high stability in terms of toxin potency, the aqueousbotulinum toxin formulation is also highly stable in terms of thesub-visible particle count. A “sub-visible particle” within the meaningof the present invention is typically a particle with a diameter below100 μm. According to the present invention, the sub-visible particlecount, more specifically the count (or number) of particles equal to orgreater than 10 μm, in the aqueous botulinum toxin formulation istypically below 1000/ml, preferably below 600/ml and more preferablybelow 200/ml during storage for 6 to 24 months (e.g., 6, 9, 12, 15, 18or 24 months) at 2-25° C. (e.g., at 5° C. or 25° C.).

Particle number measurements may be conducted by different methods, suchas Micro-Flow Imaging (MFI), Resonant Mass Measurement (RMM), andNanoparticle Tracking Analysis (NTA). The particle measurements usuallyfollow USP <788>. Within the context of the present invention, theMicro-Flow Imaging method is preferably used. This measurement methodmay, for example, be conducted using a DPA-5200 particle analyzer system(ProteinSimple, Santa Clara, Calif., USA) equipped with a silane coatedhigh-resolution 100 μm flow cell. Generally, the samples are analyzedundiluted.

Alternatively, Resonant Mass Measurements (RMM) may be employed todetermine the number of particles using, for example, the ARCHIMEDESParticle Metrology System (Affinity Biosensors, Santa Barbara, Calif.,USA) equipped with a microsensor (size range 0.3-4 μm) calibrated with 1μm polystyrene standards. All samples are typically analyzed withoutdilution. The results may be analyzed using the ParticleLab software(v1.8.570) with a size bin step of 10 nm. As another alternative fordetermining the particle numbers, Nanoparticle Tracking Analysis (NTA)may be used, for example, using a NanoSight LM20 system (NanoSight,Amesbury, UK). The samples are typically measured undiluted. Movementsof the particles in the samples may be recorded as videos for 60 secondsat ambient temperature and analyzed using suitable software (e.g., theNTA 2.3 Software).

Moreover, the aqueous botulinum toxin formulation shows high pHstability in that the pH value is essentially stable during storage ofthe prefilled syringe. Preferably, the pH value is not increased ordecreased by more than 10%, 8% or 6%, relative to the initial pH value,upon storage of the prefilled syringe for 6 to 24 months (e.g., 6, 9,12, 15, 18 or 24 months) at 2-25° C. (e.g., at 5° C. or 25° C.), forexample for 18 months at 25° C. or for 24 months at 25° C. The pH may bemeasured in accordance with the US Pharmacopeia standardized test methodUSP <791>, which outlines pH measurements for a multitude ofpharmaceutical products. Any suitable pH meter may be used, for examplethe Lab 870 pH meter of Schott Instruments.

As used herein, the term “prefilled syringe” refers to a syringe whichis filled with a drug composition (i.e. an aqueous botulinum toxinformulation) prior to distribution to the end user who will administerthe drug to the patient. The term “aqueous formulation”, as used herein,is intended to refer to an aqueous solution, suspension, dispersion oremulsion, and preferably refers to an aqueous solution. Generally, aprefilled syringe includes a drug containment container forming part ofa syringe body (i.e. a syringe barrel), a plunger to seal the proximalopening of the syringe and for expelling the drug, and a sealing device(e.g., a tip cap or a needle shield) on the outlet end of the syringe(e.g., the open end of the syringe tip or of a pre-mounted needle(cannula)) to seal the distal outlet opening. The term “prefilledplastic syringe” within the meaning of the present invention refers to aprefilled syringe, of which at least the barrel is made of plastic.

Within the present invention, the prefilled syringe is preferably a Luerslip or Luer lock syringe equipped with a tip cap (if no needle ispre-mounted) or a needle shield (if the needle is pre-mounted). Withinthe meaning of the present invention, a “luer slip syringe” is a syringethat allows a needle to be pushed on to the end of the tip, whereas a“Luer-Lock syringe” is a syringe that allows a needle to be twisted ontothe tip and then locked in place. This provides a secure connection andprevents accidental removal of the needles of the injection of fluids.

The prefilled plastic syringe according to the present invention isgenerally sterilized (e.g., by gamma radiation, ethylene oxide (ETO)treatment and moist heat (e.g., autoclaving)). The sterilization may becarried out prior to aseptic filling with the aqueous botulinum toxinformulation or after filling with the aqueous botulinum toxinformulation. The final prefilled plastic syringe is ready-to-use.Further, the prefilled syringe described herein is usually intended forsingle use and intended to be disposable. Prior to sterilization, theinner surface of the plastic syringe barrel is typically coated with alubricant to ease gliding of the plunger stopper and extruding thesyringe content.

In accordance with the present invention, the aqueous botulinum toxinformulation in the prefilled plastic syringe contains the botulinumtoxin at a concentration of, for example, 1 U/ml to 3000 U/ml or 10 U/mlto 1000 U/ml. Preferably, the botulinum toxin is present at aconcentration of about 10 U/ml to 400 U/ml, more preferably about 25U/ml to 200 U/ml, and most preferably about 40 U/ml to 150 U/ml (e.g.,50 U/ml, 75 U/ml or 100 U/ml).

The term “botulinum toxin”, as used herein, broadly refers to anyform/or type of botulinum toxin. More specifically, the botulinum toxinmay be selected from botulinum toxin types A, B, C1, C2, D, E, F, G, ormixtures thereof. Preferably, the botulinum toxin is of serotype A, B orC1, particularly of serotype A.

Furthermore, the term “botulinum toxin” is intended to include both thebotulinum toxin complex (the “toxin complex”) and the “neurotoxiccomponent” of a botulinum toxin (complex). As used herein, the term“botulinum toxin complex” or “toxin complex” refers to a high molecularweight complex comprising the neurotoxic component of approximately 150kDa and, in addition, non-toxic proteins of Clostridium botulinum,including hemagglutinin and non-hemagglutinin proteins. The botulinumtoxin serotype A complex is commercially available, for example, asBotox® (Allergan, Inc.) or as Dysport® (Ipsen, Ltd.).

The term “neurotoxic component”, as used herein, relates to theneurotoxic polypeptide of the toxin complex (the “150 kDa” polypeptide;usually in its two-chain form) without any associated non-toxicproteins. The pure neurotoxic component is, for example, commerciallyavailable under the trade names Xeomin® and Bocouture® (MerzPharmaceuticals GmbH). Preferably, the term “botulinum toxin” means theneurotoxic component of a botulinum toxin complex of a given serotype(e.g., serotype A, B or C1, particularly serotype A). In other words,the aqueous botulinum toxin formulation contained in the prefilledplastic syringe preferably contains (only) said neurotoxic component andis devoid of any other proteins of the Clostridium botulinum toxincomplex.

It is also contemplated that the present invention encompassesfunctional (i.e. biologically active) isoforms, homologs, orthologs,paralogs and fragments of botulinum toxin that show at least 50%, atleast 60%, at least 70%, at least 80%, at least 90% and up to 60%, up to70%, up to 80%, up to 90%, or up to 99% sequence identity to the aminoacid sequence of wild-type botulinum toxin, such as wild-type botulinumtoxin A or the neurotoxic component of botulinum toxin of serotype A1deposited with the GenBank database under the accession number AAA23262.The sequence identity can be calculated by any algorithm suitable toyield reliable results, for example by using the FASTA algorithm (W. R.Pearson & D. J. Lipman, PNAS 85:2444-2448, 1988). Sequence identity maybe calculated by comparing two polypeptides or two domains such as twoLC domains or fragments thereof.

Modified and recombinant botulinum toxins are also within the scope ofthe present invention. With respect to suitable mutants, reference ismade to WO 2006/027207, WO 2009/015840, WO 2006/114308, WO 2007/104567,WO 2010/022979, WO 2011/000929 and WO 2013/068476, which are allincorporated by reference herein. Furthermore, the present inventionalso refers to botulinum toxins, which are chemically modified, e.g. bypegylation, glycosylation, sulfatation, phosphorylation or any othermodification, in particular of one or more surface or solvent exposedamino acid(s). The modified, recombinant, isoforms, homologs, orthologs,paralogs, fragments, and mutants suitable for use within the presentinvention are biologically active, i.e. able to translocate into thecytosol of presynaptic cholinergic neurons and cleave proteins of theSNARE complex (e.g., VAMP/syntaxin, synaptobrevin, and SNAP-25) to exertits acetylcholine inhibitory effects.

Within the context of the present invention, the aqueous botulinum toxinformulation may comprise various other pharmaceutically acceptablesubstances, for example, salts (e.g., sodium chloride), stabilizingproteins (e.g., albumin, gelatin), sugars (e.g., glucose, fructose,galactose, trehalose, sucrose and maltose), carbohydrate polymers (e.g.,hyaluronic acid and polyvinylpyrrolidone (PVP)), polyols (e.g., glyceroland sugar alcohols like mannitol, inositol, lactitol, isomalt, xylitol,erythritol, sorbitol), amino acids, vitamins (e.g. vitamin C), zinc,magnesium, anesthetic agents (e.g., local anesthetic agents likelidocaine), surfactants, tonicity modifiers, and the like. The term“pharmaceutically acceptable”, as used herein, refers to those compoundsor substances which are suitable for contact with the tissues ofmammals, especially humans.

Furthermore, the term “comprise”, as used herein, is intended toencompass both the open-ended term “include” and the closed term“consist (of)”. The term “made of”, as used herein, is intended tobroadly relate to “produced of/from”, in particular mainly producedfrom, and generally means “comprising” (indicating that other substancesor materials may be included in some amounts). It may also mean“consisting of”.

In accordance with the present invention, the pH of the aqueousbotulinum toxin formulation in the prefilled syringe during storage ispreferably in the range of 6.0 to 7.5, 6.5 to 7.5, 6.1 to 7.3 or 6.2 to7.2, more preferably in the range of 6.3 to 7.1, and most preferably inthe range of 6.5 to 7.0. A pH within the indicated range of 6.1 to 7.3is advantageous since injections of such essentially neutral or onlyslightly acidic solutions are much less painful upon injection thanacidic solutions with a pH of below 6.

The term “aqueous formulation” or “aqueous botulinum toxin formulation”,as used herein, is not particularly limited and may refer to an aqueoussuspension, aqueous dispersion, aqueous emulsion and is preferably anaqueous solution.

Preferably, the aqueous botulinum toxin formulation preferably does notcontain a buffer like a phosphate buffer, a phosphate-citrate buffer, alactate buffer, an acetate buffer and the like. The term “buffer” asused herein denotes a pharmaceutically acceptable excipient, whichstabilizes the pH of a pharmaceutical preparation. Furthermore, theaqueous botulinum toxin formulation may be free of amino acids (e.g.,methionine) and/or surfactants (e.g., polysorbates such as polysorbate80) and/or animal-derived proteins (e.g., human serum albumin (HSA) orbovine serum albumin (BSA)).

A preferred aqueous botulinum toxin formulation for use herein compriseswater, botulinum toxin (e.g., the neurotoxic component of botulinumtoxin, preferably of serotype A) at a concentration of, e.g., 10 to 150U/ml, a salt (e.g., sodium chloride) in a concentration of, e.g., 0.5%to 1.5% w/v, a sugar (e.g., a mono- or disaccharide, such as glucose,fructose, galactose, trehalose, sucrose and maltose) at a concentrationof, e.g., 0.1% to 2% w/v, and a stabilizing protein (e.g., albumin) at aconcentration of, less than 4%, 3%, 2% or 1% w/v, e.g., 0.01% to 1% w/v.

A particularly preferred aqueous botulinum formulation for use hereinessentially consists of water, botulinum toxin (e.g., the neurotoxiccomponent of botulinum toxin type A), sodium chloride, sucrose, andalbumin (e.g., human serum albumin; HSA). The concentration of thementioned ingredients may be in the following ranges: 10 to 200 U/ml,preferably 30 to 125 U/ml (botulinum toxin); 0.5% to 1.5% w/v,preferably 0.7% to 1.1% w/v (sodium chloride); 0.1% to 2% w/v,preferably 0.2% to 1% w/v (sucrose); 0.01% to 1% w/v, preferably 0.05%to 0.5% w/v (HSA). Another particularly preferred botulinum toxinformulation for use herein is a Xeomin® solution, e.g., a Xeomin®solution reconstituted with physiological saline (0.9% sodium chloride),containing 20 to 150 U/ml of the neurotoxic component of botulinum toxintype A.

The term “essentially consists of”, as used herein is intended to meanthat substances other than those indicated are only contained in traceamounts, e.g. unavoidable impurities contained in the components usedfor formulating the aqueous botulinum toxin formulation or impuritiesincluded in the isolated botulinum toxin (e.g., the neurotoxic componentof botulinum toxin type A) in very low amounts as a result of thepurification procedure (e.g., very low residual amounts of buffers,chelating agents and the like).

In accordance with the present invention, the configuration of theprefilled plastic syringe is not particularly limited and generallycomprises a fluid-receiving barrel that, after filling, is removablycapped by a capping device to sealingly close the distal end of thesyringe (e.g., by a “tip cap” that is removed and replaced by a needleprior to use, or a sealing means like a needle shield in case of aprefilled syringe with a removable or permanent needle), and is closedat the proximal end by its plunger or any other means that is influid-tight engagement with the inner wall of the barrel. To use theprefilled syringe, the tip cap, needle shield or other type of cappingdevice is removed, optionally a needle is attached (if not alreadypresent), and the plunger tip or piston is advanced in the barrel toinject the contents (i.e. the aqueous botulinum toxin formulation) ofthe barrel into a patient.

The prefilled plastic syringe according to the present inventionpreferably comprises:

-   -   (a) a plastic syringe barrel including a proximal end and a        distal end, and a generally cylindrical wall extending        therebetween and defining a barrel lumen, the syringe barrel        having a distally projecting tip with a fluid passage extending        therethrough and communicating with the barrel lumen, wherein        the generally cylindrical wall has an interior surface        optionally coated with a barrier layer,    -   (b) a capping device having an outlet engaging portion sealingly        engaging and closing the distal open outlet end of the syringe,        wherein the outlet engaging portion is made of an elastomeric        material that optionally has a coating on its surface, and    -   (c) a plunger rod assembly which extends into the proximal end        of the syringe barrel and includes a plunger stopper in sliding        fluid-tight engagement with the cylindrical wall of the barrel        lumen, wherein the plunger stopper is made of an elastomeric        material, which optionally has a coating on at least a portion        of the plunger stopper contacting the aqueous botulinum toxin        formulation during storage and/or injection.

The materials of the prefilled syringe that have the potential tointeract with the aqueous botulinum toxin formulation in the prefilledsyringe are generally selected to minimize or limit the amount ofextractables and leachables since extractables/leachables have thepotential to contaminate the aqueous botulinum toxin formulation and toimpair the stability, e.g., in terms of biological activity or potency,of the botulinum toxin.

As used herein, the terms “extractable(s)” and “leachable(s)” refer tochemical species that can be released from a container or component ofmaterial of the prefilled plastic syringe and/or has migrated fromsyringe materials into the aqueous botulinum toxin formulation undernormal conditions of use or storage. Methods for identification ofextractables/leachables are known in the art and based on recommendedindustry practices and International Conference for Harmonisation (ICH)guidelines (see, e.g., FDA guidance, Container Closure Systems forPackaging Human Drugs and Biologics). Exemplary methods include, e.g.,Liquid Chromatography/Mass Spectrophotometry (LC/MS), Gas ChromatographySpectroscopy/Mass Spectrophotometry (GC/MS), Inductively Coupled Plasma(ICP) and Infrared (IR).

Within the context of the present invention, the inside surface of theplastic barrel may be coated or may not be coated. It is, however,usually coated with a barrier layer for lubrication purposes (in thefollowing also referred to as “lubricant layer”). The lubricant layershould not only provide high lubricity, enabling the plunger to easilyglide through the barrel, but also be compatible with the aqueousbotulinum toxin formulation and protect its shelf life. Within thecontext of the present invention, the lubricant layer may be asilicone-free lubricant layer or a silicone lubricant layer.

Likewise, the inner surface of the plastic vessel part of the vial, theinner surface of the plastic cylinder of the carpule, and the innersurface of the plastic ampoule may be optionally coated with a barrierlayer and, in particular, with a silicone-free layer or a siliconelayer. Thus, all comments provided below with regard to thesilicone-free lubricant layer and the silicone lubricant layer of theplastic syringe equally apply to the silicone-free layer and siliconelayer, respectively, of the plastic vial, plastic carpule and plasticampoule.

Suitable silicone-free fluoropolymer lubrication layers may be made ofthe materials described below for the optionally present coatings of thecapping device (or more specifically of the outlet engaging portion) andthe plunger stopper. Preferred silicone-free lubrication layers includefluoropolymer (fluorocarbon) layers, in particularethylene-tetrafluoroethylene (ETFE) layers and perfluoropolyether-based(PFPE-based) layers (e.g., TriboGlide®), as well as silicon oxide-basedglass PECVD (plasma-enhanced chemical vapor deposition) coatings.

Such fluoropolymer layers can be prepared as known in the art, forexample by spraying plastic syringe barrels with a perfluoropolyetheroil to achieve a thin layer of lubricant on the inside surface of thesyringe, followed by exposing the inner cavities to a downstream inertgas (e.g., argon or helium) plasma. The plasma treatment leads tocrosslinking of the perfluoropolyether, thereby immobilizing the coatingand reducing its tendency to migrate off the target surface, resultingin less particles that potentially impairs the stability/efficacy of thebotulinum toxin drug. An exemplary production process is described in WO2014/014641 A1, the content of which is incorporated herein byreference. Furthermore, a particularly suitable silicone-free barriercoating for use herein is known in the art as TriboGlide® coating, aperfluoropolyether coating crosslinked by plasma treatment.

A suitable silicone lubricant layer for use herein may be prepared by asiliconization method selected from, but not limited to, siliconeoil-based methods (e.g., spray-on siliconization or baked-onsiliconization) and vapor deposition methods (e.g., plasma enhancedchemical vapor deposition (PECVD)). Preferably, the silicone lubricantlayer is formed by spray-on siliconization or, more preferably, bybaked-on siliconization.

In the spray-on siliconization method, a silicone oil (e.g. DOW CORNING®360 with a viscosity of 1000 cSt) is sprayed into the syringe (i.e. thebarrel) using, e.g., a diving or static nozzle to produce a thinsilicone oil layer. While silicone oil is an excellent lubricant, excesssilicone oil can lead to the formation of unwanted visual and subvisualsilicone oil particles. With protein-based drugs, in particular, thesesilicone oil particles may lead to undesirable interactions with proteindrugs. For example, subvisual silicone oil particles are thought topromote protein aggregation. Therefore, since it results in fewersub-visual and visual silicone oil particles, the baked-onsiliconization processes is particularly preferred for use herein. Itinvolves the application of silicone oil as an emulsion (e.g., DOWCORNING® 365 siliconization emulsion), which is then baked on theplastic surface at a specific temperature and for a specific time.

The design of the syringe plastic barrel is not particularly limited andtypically has an inside diameter adjusted to accommodate the desiredfill volume of, e.g., 0.5 cm³, 1.0 cm³, 1.5 cm³ or 2.0 cm³. Usually, thesyringe barrel has graduated marks indicating the volume of fluid in thesyringe. In addition, the syringe barrel may include a flange-styleinterface. The design of the flange may, for example, be compatible withISO11040. The flange-style interface may further be compatible with anoptionally present handle.

The syringe tip is usually integrally formed (e.g., molded) with thesyringe plastic barrel. Preferably, the syringe barrel includes anintegrally formed Luer lock tip or an integrally formed Luer slip tip.The tip is formed with an integral passage extending axially through thetip and being in communication with the chamber for dispensing thecontents of the syringe barrel. The tip may have a substantiallyfrustoconical shape that converges from the distal outlet end of thesyringe barrel towards the tip's outlet end. Alternatively, the tip maybe characterized as divergent (i.e., expanding from a smaller diameterto a larger one). Furthermore, the tip is usually located centrally inrelation to the body of the syringe (concentric syringe tip) but mayalso be located offset towards the edge of the body (eccentric syringetip).

With respect to the material of the syringe plastic barrel, the plasticmaterial is preferably a cycloolefin polymer (COP), a cycloolefincopolymer (COC) or a mixture thereof. COCs are produced bypolymerization of cyclic monomers such as norbornene with ethane whileCOPs are produced by ring-opening metathesis of cyclic monomers followedby hydrogenation. The COC, COC and COP/COC materials exhibit a number ofdesirable characteristics, including high transparency, low density,excellent moisture barrier capabilities, and resistance to aqueous andpolar organic media. Specific examples include Topas® COC and DaikyoCrystal Zenith®.

The plastic vials, carpules and ampoules may be made of the plasticmaterials described above in relation to the plastic syringe barrel,polyethylene (PP, e.g., HDPE, LDPE), polyester, polyethyleneterephthalate (PET), polyamides, and mixtures thereof. It is alsocontemplated that the plastic vials, carpules and ampoules have amultilayered structure with on layer being made of one of the saidmaterials and the other layer(s) made of one (or more) other materials.

In accordance with the present invention, the “capping device” broadlyrefers to any means for closing and sealing the distal open outlet endof a syringe. Within the present invention, the term “open outlet end”or “distal open outlet end” refers to any distal open end of a syringethat is in fluid communication with the barrel lumen. The capping devicegenerally has a channel with a closed end and an open end having adimension for receiving and efficiently sealing the open outlet end ofthe syringe to prevent leakage.

In case of a prefilled plastic syringe without pre-mounted needle, thecapping device is a capping means commonly known as “tip cap”. The tipcap forms a fluid-tight seal with the tip of the syringe to efficientlyclose the syringe barrel and to prevent leakage of the contents of thesyringe barrel. The tip cap is usually removable coupled to the syringetip or a luer collar. The luer collar surrounds the top of the syringebarrel (e.g., syringe tip). Preferably, the luer collar has internalthreads and the tip cap has external threads complementing said internalthreads of said luer collar for coupling the tip cap to the syringebarrel. In case of the prefilled plastic syringe of the presentinvention, the luer collar is generally integrally formed (e.g.,unitarily molded with) the syringe barrel. Prior to use, the tip can beremoved, and a needle cannula (needle assembly) can then be securelycoupled to the syringe tip.

In case the prefilled plastic syringe includes a removable ornon-removable (i.e. permanent) cannula or needle cannula (also referredto as “needle” or “needle assembly”) extending from the syringe tip fordelivering the aqueous botulinum toxin formulation from said syringe,the capping device may be referred to as “needle shield”. Said needleshield generally has a channel with a closed end and an open end havinga dimension for receiving and coupling with the cannula (needle) mountedon the tip of the syringe. Typically, the (sharpened) end of the cannulapenetrates the closed end of the channel in the needle shield to sealthe open end of the cannula.

The capping device (e.g., tip cap or needle shield) may be a unitarymember and usually made from a flexible and resilient polymeric material(e.g., an elastomer), or can have an outer cap made of a rigid plasticmaterial that is coupled to a flexible and resilient inner cap ormaterial comprising, or made of, e.g., an elastomer, at least a portionof which contacts and seals the distal opening of the syringe.Generally, at least an outlet engaging portion that contacts the distaltip opening to form a fluid-tight seal is made from a flexible and/orresilient material (e.g., an elastomer) and, since the engaging portioncontacts the aqueous botulinum toxin formulation during storage and/oruse, is preferably made of a material having a minimized potential forunwanted extractables/leachables. In order to further decrease theamount of extractables and/or leachables and to increase compatibilitywith the aqueous botulinum toxin formulation, the outlet engagingportion may have a coating thereon.

Suitable flexible and/or resilient materials of the capping device, inparticular of the outlet engaging portion, include elastomers that donot interfere with the aqueous botulinum toxin formulation and enablelong-term storage. In particular, the part of the sealing device thatcontacts, or is configured to contact, the aqueous botulinum toxinformulation (i.e. the outlet engaging portion) should exhibit lowextractable/leachable levels during prolonged storage of the aqueousbotulinum toxin formulation. As used herein, the term “elastomer” or“elastomeric material” refers primarily to crosslinked thermosettingrubbery polymers that are more easily deformable than plastics but thatare approved for use with pharmaceutical grade fluids and are notreadily susceptible to leaching or gas migration.

Preferably, the elastomeric material suitable for use herein is selectedfrom isoprene rubber (IS), butadiene rubber (polybutadiene, BR), butylrubber (copolymer of isobutylene and isoprene; IIR), halogenated butylrubber (e.g., chloro butyl rubber, CIIR; and bromo butyl rubber: BIIR),styrene-butadiene rubber (copolymer of styrene and butadiene, SBR), andmixtures thereof. Preferably, the elastomeric material is butyl rubberor a halogenated butyl rubber, particularly a bromo butyl rubber or achloro butyl rubber, or a mixture thereof. The elastomeric material mayalso be reinforced with an inert mineral. Further, it may be cured(e.g., with organic peroxide, phenolic resins, etc.).

Suitable coatings that may be optionally present on the outlet engagingportion made from, e.g., the above-mentioned elastomeric materials, aregenerally made of a material that does not undesirably interfere withthe aqueous botulinum toxin formulation and exhibits low levels ofextractables/leachables. Coatings for use herein include, but are notlimited to, polypropylene, polyethylene, parylene (e.g., parylene N,parylene C and parylene HT), crosslinked silicone and, preferably,fluoropolymer coatings. Examples of suitable crosslinked siliconecoatings include the B2-coating (Daikyo Seiko) or XSi™ (BectonDickinson).

The fluoropolymer coatings include, but are not limited to, fluorinatedethylene-propylene copolymers (e.g.,tetrafluoroethylene-hexafluoropropylene copolymer (FEP)), fluorinatedethylene-ethylene copolymers (e.g., ethylene tetrafluoroethylenecopolymer (ETFE), such as FluroTec®), PVA (a copolymer oftetrafluoroethylene (TFE) and perfluoropropylvinylether (PPVE)),tetrafluoroethylene-perfluoroethylene copolymers, polyvinylidenefluoride (PVDF), polyvinyl fluoride (PVF), polytetrafluoroethylene(PTFE), and mixtures thereof. Preferably, the coating is made of ETFEand, particularly, is a FluroTec® coating.

With regard to the carpule of the present invention, the distal end issealed by a puncture membrane. The puncture membrane may be formed froma thin rubber or silicone, a thin plastic/polymer, a film such as Mylar,a polyolefin such as polyethylene or polypropylene, a metal foil such analuminum foil, etc. The membrane may be between about 0.001 and 2.0 mm,usually between 0.002 mm and 0.65 mm thick. Also, the membrane may bemade of an elastomeric material and optionally have a coating asdescribed above in connection with the capping device of the prefilledplastic syringe.

With regard to the vial of the present invention, the vial closuresystem (e.g., cap), in particular those portions of the vial closuresystem that come into contact with, or have the potential to come intocontact with and/or seal the vial (e.g., a septum) may be made of anelastomeric material, in particular a thermoplastic elastomericmaterial, more particularly a styrenic block copolymer thermoplasticelastomer, or of an elastomeric material as described above inconnection with the capping device of the prefilled plastic syringe ofthe present invention. Another suitable material is a silicone material.Furthermore, the said materials may have an optional coating, inparticular a fluoropolymer coating, as defined above in relation to thecapping device of the prefilled plastic syringe.

In accordance with the present invention, the prefilled syringegenerally includes a plunger rod assembly, which extends into theproximal end of the syringe barrel. The plunger rod assembly may includea rod (also known as pushrod) with a plunger stopper at its tip (alsoknown as “plunger”) in sliding fluid-tight engagement with thecylindrical wall of the barrel lumen. The plunger forms the proximalseal and the dynamic seal that allows for extrusion of the liquidbotulinum toxin formulation. The plunger stopper contacts the aqueousbotulinum toxin formulation during storage and/or administration.Therefore, the plunger stopper should be compatible with the aqueousbotulinum toxin formulation and not impair its long-term stability. Inparticular, the plunger stopper should preferably be designed tominimize the amount of extractables/leachables upon long-time storage.

Within the present invention, the plunger stopper is preferably made ofan elastomeric material, which optionally has a coating on at least aportion of the plunger stopper that contacts, or is capable ofcontacting, the aqueous botulinum toxin formulation during storageand/or use. Suitable plunger stopper elastomeric materials for useherein include, but are not limited to, isoprene rubber (IS), butadienerubber (polybutadiene, BR), butyl rubber (copolymer of isobutylene andisoprene, IIR), halogenated butyl rubber (e.g., chloro butyl rubber,CIIR; and bromo butyl rubber, BIIR), styrene-butadiene rubber (copolymerof styrene and butadiene, SBR), and mixtures thereof. Preferably, theplunger stopper material is a butyl rubber or a halogenated butyl rubberor a mixture thereof, particularly a bromo butyl rubber or a chlorobutyl rubber. The elastomeric material may also be reinforced with aninert mineral. Further, it may be cured (e.g., with organic peroxide,phenolic resins, etc.).

Preferably, the plunger stopper comprises a coating acting as a barrierfilm. The coating is usually applied to at least the seal surfaces,including the surface portion of the plunger stopper facing the barrellumen and contacting the aqueous botulinum toxin formulation duringstorage and/or use. The coating serves the purpose of providing goodlubricity while minimizing interaction between the plunger stopper andthe liquid botulinum toxin formulation.

Suitable coatings of the plunger stopper are generally made of amaterial that does not undesirably interfere with the aqueous botulinumtoxin formulation and exhibits low levels of extractables/leachables.Such coatings include, but are not limited to, polypropylene,polyethylene, parylene (e.g., parylene N, parylene C and parylene HT),crosslinked silicone and, preferably, fluoropolymer coatings. Examplesof suitable crosslinked silicone coatings include the B2-coating (DaikyoSeiko) or XSi™ (Becton Dickinson).

The fluoropolymer coatings include, but are not limited to, fluorinatedethylene-propylene copolymers (e.g.,tetrafluoroethylene-hexafluoropropylene copolymer (FEP)), fluorinatedethylene-ethylene copolymers (e.g., ethylene tetrafluoroethylenecopolymer (ETFE), such as FluroTec®), PVA (a copolymer oftetrafluoroethylene (TFE) and perfluoropropylvinylether (PPVE)),tetrafluoroethylene-perfluoroethylene copolymers, polyvinylidenefluoride (PVDF), polyvinyl fluoride (PVF), polytetrafluoroethylene(PTFE), and mixtures thereof. Preferably, the coating is made of ETFEand, particularly, is a FluroTec® coating.

The design of the plunger stopper is not particularly limited and may bea nested or bagged stopper. Further, the interface to the rod may bethreaded to allow installation of the rod after sterilization.Alternatively, the interface to the rod may be designed with a snap-ondesign. The rod, like the plunger stopper, is generally designed towithstand sterilization but is not otherwise limited in any particularway. Typically, the rod is made of a plastic material such as anethylene vinyl acetate (EVA) copolymer or polypropylene.

The rubber stopper of the carpule of the present invention may comprise,or be made of, the same elastomeric materials as described above inconnection with the plunger stopper of the plastic syringe. Also, therubber stopper of the carpule may have the same optional coating asdefined above with respect to the coating on the plunger stopper.Further, the coating may be on at least a portion of the rubber stopperthat contacts the aqueous botulinum toxin formulation during storageand/or use.

Within the framework of the present invention, the prefilled plasticsyringe including the capping device, the syringe barrel, and theplunger assembly before and after sterilization (e.g., by gammaradiation, ethylene oxide or autoclaving) meet or exceed the standardsfor extractable substances as determined by The Japanese Pharmacopoeia,14th Edition, No. 61, Test Methods for Plastic Containers (2001) as wellas the standards of The Japanese Pharmacopoeia, 14th Edition, No. 59,Test for Rubber Closure for Aqueous Infusions. Furthermore, the polymercomposition of the capping device and plunger stopper aftersterilization satisfies the combustion tests of No. 61 of The JapanesePharmacopoeia, Test Methods for Plastic Containers (2001), as well asthe acceptable limits for extractable substances as defined by thefoaming test, pH test, potassium permanganate-reducing substances test,UV spectrum test and residue on evaporation test according to TheJapanese Pharmacopoeia, No. 61, Test Methods for Plastic Containers(2001).

In another aspect, the present invention relates to a kit comprising aprefilled plastic container (e.g., a syringe, vial, carpule or ampoule)according to the present invention and, optionally, instructions for useof said prefilled plastic container.

In a further aspect, the present invention relates to a prefilledplastic container (e.g., a syringe, vial, carpule or ampoule) accordingto the present invention for use in therapy. In particular, theprefilled plastic container according to the present invention may beused in the treatment of a disease or condition caused by or associatedwith hyperactive cholinergic innervation of muscles or exocrine glandsin a patient.

Within the context of the present invention, if the container is not asyringe (e.g., a vial, carpule or ampoule), the content of these“non-syringe type” containers (i.e. the aqueous botulinum toxinformulation) is generally injected to the desired target site using asuitable injection device (e.g., a syringe) in the same manner asdescribed herein in relation to the prefilled plastic syringe. Thecarpules are inserted into a carpule injection device as known to thoseskilled in the art. The contents of the vials and ampoules are generallyaseptically filled into a syringe and then injected to the target siteusing a suitable injection device (e.g., a syringe) in the same manneras described herein in relation to the prefilled plastic syringe.

The term “hyperactive cholinergic innervation”, as used herein, relatesto a synapse, which is characterized by an unusually high amount ofacetylcholine release into the synaptic cleft. “Unusually high” relatesto an increase of, e.g., up to 25%, up to 50% or more with respect to areference activity which may be obtained, for example, by comparing therelease with the release at a synapse of the same type but which is notin a hyperactive state, wherein muscle dystonia may be indicative of thehyperactive state. “Up to 25%” means, for example, about 1% to about25%. Methods for performing the required measurements are known in theart.

Within the present invention, the disease or condition caused by orassociated with hyperactive cholinergic innervation of muscles includes,but is not limited to, dystonias (e.g., blepharospasm, spasmodictorticollis, limb dystonia, and task-specific dystonias such as writer'scramps), spasticities (e.g., post-stroke spasticity, spasticity causedby cerebral palsy), paratonia, diskinesias (e.g., tardive diskinesia),focal spasms (e.g., hemifacial spasm), (juvenile) cerebral palsy (e.g.,spastic, dyskinetic or ataxic cerebral palsy), strabismus, pain (e.g.neuropathic pain), wound healing, tremors, tics, and migraine.

The prefilled botulinum toxin container (e.g., a syringe, vial, carpuleor ampoule) of the present invention is particularly useful in thetreatment of dystonia of a muscle. Exemplary dystonias include, but arenot limited to, dystonias selected from the group consisting of (1)cranial dystonia, including blepharospasm and oromandibular dystonia ofthe jaw opening or jaw closing type, (2) cervical dystonia, includingantecollis, retrocollis, laterocollis and torticollis, (3) pharyngealdystonia, (4) laryngeal dystonia, including spasmodic dysphonia, (5)limb dystonia, including arm dystonia such as task specific dystonias(e.g., writer's cramp), leg dystonia, axial dystonia, segmentaldystonia, and (6) other dystonias.

The term “hyperactive exocrine gland”, as used herein, is notparticularly limited and covers any exocrine gland with hyperactivity.It is therefore envisaged that the present invention can be applied tothe treatment involving any of the glands mentioned in Sobotta,Johannes, Atlas der Anatomie des Menschen, 22. Auflage, Band 1 and 2,Urban & Fischer, 2005, which is incorporated herein by reference.Preferably, the hyperactive gland is an autonomic exocrine gland. Thebotulinum toxin composition is preferably injected into or in thevicinity of the hyperactive exocrine gland.

Within the present invention, the hyperactive exocrine glands include,but are not limited to, sweat gland, tear gland, salivary gland, andmucosal gland. Furthermore, the hyperactive gland may also be may beassociated with a disease or condition selected from the groupconsisting of Frey syndrome, Crocodile tears syndrome, axillarhyperhidrosis, palmar hyperhidrosis, plantar hyperhidrosis,hyperhidrosis of the head and neck, hyperhidrosis of the body,rhinorrhea, or relative hypersalivation in patients with stroke,Parkinson's disease or amyotrophic lateral sclerosis. In particular, thedisease or condition caused by or associated with hyperactivecholinergic innervation of exocrine glands may include drooling(hypersalivation, sialorrhea) and excessive sweating (hyperhidrosis).

The administration is not limited to any particular administrationregimen, mode, form, dose and interval. As known to those skilled in theart, the administered amount or dose of botulinum toxin depends on themode of application, the type of disease, the patient's weight, age, sexand state of health, and which target tissues are chosen for injection.The botulinum toxin formulation is usually administered locally, e.g.,by subcutaneous or intramuscular injection into or in the vicinity ofthe target tissues (e.g., muscles, skin, exocrine glands).

Different muscles, depending on their size, generally require differentdosing. A suitable dose may range from 10 to 2000 U, preferably from 50to 500 U, and more preferably from 100 to 350 U of botulinum toxin. Forthe treatment of exocrine glands, the dose is usually in the range of 10to 500 U, preferably 20 to 200 U, and more preferably 30 to 100 U. Suchtotal amounts may be administered on the same day or on a subsequent dayof treatment. For example, during a first treatment session a firstfraction of the dose may be administered. During one or more treatmentsessions, the remaining fraction of the total dose may be administered.Further, the frequency of application is not particularly limited andsuitable administration intervals may be three months or less (e.g., 4or 8 weeks) or more than three months.

In still another aspect, the present invention relates to the use of theprefilled plastic container (e.g., a syringe, vial, carpule or ampoule)according to the present invention in cosmetic applications, such as fortreating facial asymmetries and wrinkles/lines of the skin (e.g., faciallines and facial wrinkles), including upper facial rhytides, platysmabands, glabellar frown lines, horizontal forehead lines, nasolabialfolds, chin folds, popply chin, mental ceases, marionette lines, buccalcommissures, perioral wrinkles, crow's feet, and jawlines. Preferably,the prefilled botulinum toxin container (e.g., a syringe, vial, carpuleor ampoule) of the present invention is used for injection intoglabellar frown lines, crow's feet, perioral wrinkles, and/or platysmabands.

The amounts of botulinum toxin administered for cosmetic application areusually in the range of 1 to 5 U, 5 to 10 U, 10 to 20 U or 20 to 50 U.Such total amounts may be administered on the same day or on asubsequent day of treatment. For example, during a first treatmentsession a first fraction of the dose may be administered. This firstfraction is preferably a suboptimal fraction, i.e. a fraction, whichdoes not remove the wrinkles or skin lines completely. During one ormore treatment sessions, the remaining fraction of the total dose may beadministered. Regarding further details of administration, it isreferred to the disclosure provided above in relation to the therapeuticuse.

In a yet further aspect, the present invention relates to a method oftreating a disease or condition caused by or associated with hyperactivecholinergic innervation of muscles or exocrine glands in a patient, themethod comprising locally administering an effective amount of botulinumtoxin to a muscle or exocrine gland of the patient using the prefilledplastic container (e.g., a syringe, vial, carpule or ampoule) accordingto the present invention.

As used herein, the term “effective amount” refers to the amount of abotulinum toxin sufficient to effect beneficial or desired therapeutic,cosmetic or anesthetic results. The term “patient”, as used herein,generally relates to a human afflicted with a disease or conditioncaused by or associated with hyperactive cholinergic innervation ofmuscles or exocrine glands, or to a human in need of a cosmetic oranesthetic treatment. As used herein, “patient” may be interchangeablyused with “subject” or “individual”.

The term “local administration” within the meaning of the presentinvention refers preferably to subcutaneous or intramuscular injectioninto or in the vicinity of the target tissues (e.g., muscles, skin,exocrine glands). With respect to the administration (e.g., regimen,mode, form, dose and interval) and the disease or conditions to betreated, the same comments apply as those set out above in relation tothe use of the glass container (e.g., the prefilled botulinum toxinsyringe) for cosmetic and therapeutic applications.

In a still further aspect, the present invention relates to a method forthe cosmetic treatment of the skin, such as for treating wrinkles of theskin and facial asymmetries, the method comprising locally administeringan effective amount of botulinum toxin to a patient by intradermal,subdermal or subcutaneous injection using the prefilled plasticcontainer (e.g., a syringe, vial, carpule or ampoule) according to thepresent invention.

Exemplary cosmetic applications include those mentioned above. Withregard to the meaning or definition of the terms “effective amount”,“patient”, the administration (e.g., regimen, mode, form, dose andinterval), and the disease or conditions to be treated, the commentsprovided above with regard to other aspects of the present inventionsimilarly apply, unless otherwise stated.

The present invention will now be further illustrated by the following,non-limiting examples.

Examples

The following examples show that, contrary to expectation and commonbelief in the art, an aqueous botulinum toxin formulation stored in aprefilled syringe system exhibits an excellent stability for a prolongedtime period (e.g., for 9-12 months) at standard refrigerator temperature(2-8° C.). Furthermore, extrapolation of the measured stability dataindicates that the prefilled botulinum toxin syringe is highly stablefor at least 12 to 24 months at 2-8° C.

Accordingly, the botulinum toxin's presentation can be changed from alyophilized vial to a prefilled plastic syringe format, which meets thedemands of physicians and patients looking for easier, safer and moreaccurate modes of administration.

Materials & Methods

An aqueous botulinum liquid botulinum toxin formulation was prepared bydissolving 1.0 mg human serum albumin (HSA), 4.7 mg sucrose, andincobotulinum-toxinA in 0.9% saline to a concentration of 50 U/ml. Theformulation was then filled into a syringe plastic barrel with aLuer-Lock-type closure comprising a Luer-Lock and a tip cap which, whenfitted, contacts the opening of the distal syringe tip in order to sealthe syringe barrel. Afterwards, a plunger stopper was inserted into theproximal end portion of the barrel in order to close the proximalopening. The resulting prefilled plastic syringe was then stored at atemperatures of 5° C. or 25° C. Then, the stability of the aqueousbotulinum toxin formulation at t=0, 1, 3, 6, 9 and 12 months wasassessed by determining the remaining toxin potency, pH value, andsub-visible particle count.

The toxin potency was determined using a mouse hemidiaphragm assay (HDA)according to Goschel et al. (Exp. Neurol. 147:96-102, 1997). In brief,the assay was conducted by maintaining a murine nerve muscle preparationin an organ bath containing 4 ml of medium. The muscle was attached to aforce transducer and electrically stimulated via the phrenic nerveresulting in an isometric contraction force which remained constant formore than 180 min if no toxin was added. Upon introduction of toxin tothe organ bath, the contraction amplitude of the nerve-stimulated musclegradually declines. The contraction amplitude of the diaphragm wasmonitored over time. As a read-out, the time at which half the initialcontraction force is reached was determined and referred to as“paralysis time”. Increased time values, compared to initial values,reflect lower amounts of active toxin and loss of toxin potency,respectively.

The pH measurements were performed in accordance with the USPharmacopeia standardized test method USP <791>, which outlines pHmeasurements for a multitude of pharmaceutical products, using a pHmeter (Lab 870, Schott Instruments).

Particle measurements were conducted using Micro-Flow Imaging (MFI) bymeans of a DPA-5200 particle analyzer system (ProteinSimple, SantaClara, Calif., USA) equipped with a silane coated high-resolution 100 μmflow cell. The samples were analyzed undiluted. MFI View System Software(MVSS) version 2-R2-6.1.20.1915 was used to perform the measurements,and MFI View Analysis Suite (MVAS) software version 1.3.0.1007 was usedto analyze the samples.

Two different prefilled plastic syringe systems (in the following“configurations A and B”), which differ from each other by the plungerstopper, were studied. Details of the syringe configurations examinedare summarized in Table 1.

TABLE 1 Syringe configurations A and B SYRINGE BARREL TIP CAP PLUNGERSTOPPER Product Product Product CONF. COMP. Name Material Name MaterialName Material A GH¹ ClearJect Cyclo-olefin Tip Cap 4 Sumitomo P-134 1 mLPi1 Sumitomo P-134 1 ml long polymer (Sumitomo) (chlorinated (Sumitomo)(chlorinated butyl LL2 T4 (COP); barrel butyl rubber)³ rubber; coated(Taisei is siliconized^(2,3) with cross-linked Kako) silicone)³ B GH¹See con- See con- See con- See con- West ® Elastomer figurationfiguration figuration figuration 4023/50G formulation A A A A NovaPure ®(bromobutyl reinforced with inert mineral) coated with FluroTec ® film¹= Gerresheimer ²= siliconized with Dow Corning Medical Fluid 360(viscosity 12,500 cSt) ³= sterilized by gamma-irradiation in accordancewith ISO 11137

Results

The results of the stability measurements in terms of remaining toxinpotency for configurations A and B are shown in Table 2 below.

TABLE 2 Stability in terms of potency STABILITY (toxin potency in %,relative to initial toxin activity) Time (months) CON- Temper- t = 0FIG. ature (initial)* 1 3 6 9 12 18 A 2-8° C. 100 109 106 94 94 91 106 B100 116 114 102 106 88 100 A  25° C. 100 94 85 65 0 n.d.** n.d. B 100116 78 54 0 n.d.  n.d. *initial absolute toxin activity in units rangedfrom 50 U to 54 U **n.d. = not determined

As is evident from Table 2, the toxin essentially maintains its initialpotency at 2-8° C. over time, i.e. there is essentially no potency lossafter storage for no less than 18 months (a potency loss of <10% afterstorage for 18 months). Even at room temperature (i.e., 25° C.), thestability is still acceptable, as indicated by a potency loss of no morethan about 20% after 3 months.

An extrapolation of the stability data for configurations A and B at2-8° C. up to a storage time of 24 months is graphically shown inFIG. 1. As can be seen, the estimated maximum loss of biologicalactivity after 24 months is expected to be about 10% and, thus, to beessentially the same as the loss of biological activity measured after12 months.

Furthermore, the pH measurements revealed that the pH value remainedexceptionally stable over a period of up to 18 months. No trend towardshigher or lower values was observed and all measured pH values remainedwithin ±0.4 of the initial pH (see Table 3).

TABLE 3 Stability in terms of pH STABILITY (pH) Time (months) CON-Temper- t = 0 FIG. ature (initial) 1 3 6 9 12 18 A 2-8° C. 6.8 6.8 6.86.9 6.7 n.d. 6.5 B 6.5 6.9 6.9 6.8 6.9 6.6 n.d. A  25° C. 6.8 6.7 6.76.8 6.6 6.7 n.d. B 6.5 6.1 6.8 6.7 6.9 n.d. n.d.

Moreover, the particle size measurements by Micro-Flow Imaging showed alow overall number of particles and no significant increase in thenumber of particles (see Table 4).

TABLE 4 Stability in terms of sub-visible particle count STABILITY(sub-visible particle count (equal to or greater than 10 μm)) Time(months) CON- Temper- t = 0 FIG. ature (initial) 1 3 6 9 12 18 A 2-8° C.64 139 28 148 154 91 265 B 472 126 100 90 369 97 — A  25° C. 64 192 60210 163 280 — B 472 243 81 822 366 100 —

As can be seen from Table 4, the particle counts stay well below 1000/mland in most cases even below 250/ml. Likewise, particle measurements bymeans of the Resonant Mass Measurement (RMM) method (using theARCHIMEDES particle methodology system; Affinity Biosensors, SantaBarbara, Calif., USA) and the Nanoparticle Tracking Analysis (using aNanoSight LM20 system; NanoSight, Amesbury, UK) lead to similar resultsand revealed no significant particle counts.

In conclusion, the results presented above show that liquid botulinumtoxin formulations in prefilled plastic syringes are stable over aprolonged storage time (e.g., 12-24 months) at temperatures of 2-8° C.This finding was very surprising in view of the labile nature ofbotulinum toxin, which is known to be highly heat-labile and unstable atalkaline pH. This was all the more surprising since the botulinum toxinconcentrations in prefilled syringes are exceptionally low and, thus,smallest absolute losses in the amount of active toxin will lead tolarge percentage changes.

Accordingly, the above results show that botulinum toxin can beformulated in a prefilled plastic syringe format, which offersadvantages over glass syringes in terms of break resistance, decreasedweight, increased flexibility for novel shapes of primary containers,improved dimensional tolerances, and absence of undesirable substances(e.g., adhesives). Moreover, compared to other botulinum toxinpresentations, the prefilled syringe format enhances convenience andease of handling, reduces medication errors, improves dosing accuracy,minimizes the risk of contamination, improves sterility assurance, andincreases safety in administration.

The invention claimed is:
 1. A prefilled plastic syringe comprising anaqueous botulinum toxin formulation, the plastic syringe comprising (a)a plastic syringe barrel including a proximal end and a distal end, anda cylindrical wall extending therebetween and defining a barrel lumen,the syringe barrel having a distally projecting tip with a fluid passageextending therethrough and communicating with the barrel lumen, whereinthe cylindrical wall has an interior surface optionally coated with abarrier layer, (b) a capping device having an outlet engaging portionsealingly engaging and closing the distal open outlet end of thesyringe, wherein the outlet engaging portion is made of an elastomericmaterial that optionally has a coating on its surface, and (c) a plungerrod assembly which extends into the proximal end of the syringe barreland includes a plunger stopper in sliding fluid-tight engagement withthe cylindrical wall of the barrel lumen, wherein the plunger stopper ismade of an elastomeric material, which optionally has a coating on atleast a portion of the plunger stopper contacting the aqueous botulinumtoxin formulation during storage and/or injection, wherein theelastomeric material of the outlet engaging portion is selected from thegroup consisting of isoprene rubber, butadiene rubber, butyl rubber,halogenated butyl rubber, and styrene-butadiene rubber, and mixturesthereof, and the elastomeric material of the plunger stopper is selectedfrom the group consisting of isoprene rubber, butadiene rubber, butylrubber, halogenated butyl rubber, and styrene-butadiene rubber, andmixtures thereof, and wherein the toxin activity is not reduced by morethan 25%, relative to the initial toxin activity, upon storage of theprefilled plastic syringe for 12 months at 5° C. or 3 months at 25° C.2. The prefilled plastic syringe of claim 1, wherein the number ofsub-visible particles of equal to or greater than 10 μm is below 1000/mlduring storage for 6 to 24 months at 2° C. to 25° C.
 3. The prefilledplastic syringe of claim 1, wherein the pH value is not increased ordecreased by more than 10%, relative to the initial pH value, duringstorage of the prefilled plastic syringe for 6 to 24 months at 2° C. to25° C., or wherein the pH of the aqueous botulinum toxin formulationduring storage is maintained in the range of 6.1 to 7.3, or both.
 4. Theprefilled plastic syringe of claim 1, wherein the botulinum toxin ispresent in the aqueous formulation at a concentration of 10 U/ml to 1000U/ml.
 5. The prefilled plastic syringe of claim 1, wherein the aqueousbotulinum toxin formulation in the prefilled plastic syringe does notcontain a buffer.
 6. The prefilled plastic syringe of claim 1, whereinthe barrier layer of the syringe barrel is present and is asilicone-free layer or a silicone layer.
 7. The prefilled plasticsyringe of claim 1, wherein the optional coating on the outlet engagingportion is present and is a crosslinked silicone coating or afluoropolymer coating.
 8. A kit comprising a prefilled plastic syringe,wherein said prefilled plastic syringe comprises an aqueous botulinumtoxin formulation, the plastic syringe comprising (a) a plastic syringebarrel including a proximal end and a distal end, and a cylindrical wallextending therebetween and defining a barrel lumen, the syringe barrelhaving a distally projecting tip with a fluid passage extendingtherethrough and communicating with the barrel lumen, wherein thecylindrical wall has an interior surface optionally coated with abarrier layer, (b) a capping device having an outlet engaging portionsealingly engaging and closing the distal open outlet end of thesyringe, wherein the outlet engaging portion is made of an elastomericmaterial that optionally has a coating on its surface, and (c) a plungerrod assembly which extends into the proximal end of the syringe barreland includes a plunger stopper in sliding fluid-tight engagement withthe cylindrical wall of the barrel lumen, wherein the plunger stopper ismade of an elastomeric material, which optionally has a coating on atleast a portion of the plunger stopper contacting the aqueous botulinumtoxin formulation during storage and/or injection, wherein theelastomeric material of the outlet engaging portion is selected from thegroup consisting of isoprene rubber, butadiene rubber, butyl rubber,halogenated butyl rubber, and styrene-butadiene rubber, and mixturesthereof, and the elastomeric material of the plunger stopper is selectedfrom the group consisting of isoprene rubber, butadiene rubber, butylrubber, halogenated butyl rubber, and styrene-butadiene rubber, andmixtures thereof, and wherein the toxin activity is not reduced by morethan 25%, relative to the initial toxin activity, upon storage of theprefilled plastic syringe for 12 months at 5° C. or 3 months at 25° C.9. A method for the treatment of a disease or condition caused by orassociated with hyperactive cholinergic innervation of muscles orexocrine glands in a patient, the method comprising locallyadministering an effective amount of botulinum toxin to a muscle orexocrine gland of the patient by injection using the prefilled plasticsyringe according to claim
 1. 10. A method for the cosmetic treatment ofthe skin, optionally for treating wrinkles of the skin and facialasymmetries, the method comprising locally administering an effectiveamount of botulinum toxin to a patient by intradermal, subdermal orsubcutaneous injection using the prefilled plastic syringe according toclaim
 1. 11. The prefilled plastic syringe of claim 1, wherein theoptional coating on the plunger stopper is present and is a crosslinkedsilicone coating or a fluoropolymer coating.
 12. The method of claim 9,wherein the disease or condition caused by or associated withhyperactive cholinergic innervation of muscles or exocrine glands isselected from the group consisting of dystonia, spasticity, paratonia,diskinesia, focal spasm, strabismus, tremor, tics, migraine, sialorrhea,and hyperhidrosis.
 13. The plastic prefilled syringe according to claim1, wherein the cylindrical wall has an interior surface coated with abarrier layer.
 14. The method according to claim 10, comprising treatingwrinkles of the skin and facial asymmetries.
 15. A kit according toclaim 8, further comprising instructions for use of said prefilledplastic syringe.